Cell lines and synchronization
U2OS cells and HNFs, either wild type or expressing the viral oncoprotein E6 (gift from J. Piette and V. Gire, Centre de Recherche en Biochimie Macromoléculaire, Montpellier, France), were grown in DME with 10% FBS (Invitrogen) supplemented with penicillin and streptomycin. Synchronization of U2OS cells at the G1/S boundary was performed by a double thymidine block. Cells were first blocked with 2.5 mM thymidine for 15 h, released for 8 h, and then blocked again with 2.5 mM thymidine for 15 h. To control the efficiency of synchronization, flow cytometry analysis was performed.
shRNA preparation and transfection
A human PR-Set7 shRNA directed against the PR-Set7 mRNA sequence GATGCAACTAGAGAGACA (nucleotides 854–871) was checked against the human genome sequence to ensure that only the PR-Set7 mRNA would be targeted. A firefly luciferase nonspecific control shRNA was used. The shRNA sequences were introduced into the puromycin retroviral vector RNAi Ready pSiren (BD Biosciences) according to the manufacturer's instructions. Cells were infected with the corresponding retroviral particles as described previously (Le Cam et al., 2006
) and were selected 24 h later in 10 μg/ml puromycin.
Mutagenesis and generation of PR-Set7SR cell lines
A hygromycin retroviral vector pMSCV was modified to encode the HA epitope tag downstream of the ATG, creating pMSCV-HA vector. The HA epitope tag sequence is followed by an SpeI–BamH1 polylinker. Spe1–BamH1 cDNA fragments encoding the different PR-Set7 truncations were cloned into pMSCV-HA vector. The PR-Set7SR
constructs were prepared by introducing five silent mutations into PR-Set7 cDNA as described previously (Julien and Herr, 2003
). For stable PR-Set7SR
expression, U2OS cells were infected as described previously (Le Cam et al., 2006
) and selected with 60 μg/ml hygromycin B.
Cells were incubated with 300 μM BrdU (Sigma-Aldrich) for 1 h, fixed with a 70% ethanol solution, and permeabilized with 0.2% Triton X-100 for 10 min. Then, cells were treated with 0.2 N HCl before staining with mouse antibody to BrdU (1:30 diluted in PBS with 0.2% Tween 20 and 1% BSA; Becton Dickinson) for 1 h at room temperature followed by 1-h incubation with an FITC-conjugated antibody (1:300; BD Biosciences). DNA was then counterstained by overnight incubation with 7-amino-actinomycin D (7AAD; 1:50; Sigma-Aldrich) in the presence of RNase. Cell cycle profiles were analyzed by a flow cytometer (FACScan; Becton Dickinson) using CellQuest software (Becton Dickinson).
Western blot analysis
For immunoblot analysis, cells washed with PBS were lysed in laemmli buffer (for histones, acid extraction was performed according to the Abcam protocol). After measuring protein quantity by Bradford, equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane (Whatman), and probed with one of the following antibodies: mouse antiphospho-ATM-Ser1981 (1:1,000; Cell Signaling Technology), rabbit antiphospho-ATR-Ser428 (1:1,000; Cell Signaling Technology), rabbit antiphospho-Chk1-Ser345 (1:1,000; Cell Signaling Technology), mouse anti-Chk1 (1:1,000; gift from V. Gire), rabbit anti-p21 (1:500; gift from V. Dulik, Institut de Génétique Moléculaire, Montpellier, France), rabbit antiphospho-p53-Ser15 (1:500; gift from V. Gire), mouse anti-p53 (1:1,000; gift from V. Gire), rabbit anti–PR-Set7 (1:1,000; Millipore), mouse anti–β-actin (1:20,000; Sigma-Aldrich), mouse antiphospho-H2A.X-Ser139 (1:1,000; Millipore), rabbit antiphospho-histone H3-Ser10 (1:1,000; Millipore), rabbit anti–H4-K20-1xMe, -2xMe, or -3xMe (1:1,000; Millipore), rabbit anti–histone H4 (1:1,000; Millipore), and mouse anti-HA tag (12CA5; Cold Spring Harbor Laboratory). Membranes were then incubated with the appropriate HRP-conjugated secondary antibodies. The immunoreactive bands were detected by chemiluminescence (Thermo Fisher Scientific).
For immunofluorescence, shRNA-infected cells grown on glass coverslips were fixed in 3% PFA for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min, and blocked for 1 h with 1% BSA (Roche) at room temperature. To visualize PR-Set7, cells on coverslips were pretreated with 15 μM MG132 for 1 h and incubated for 10 min at 4°C with a solution of PBS and 0.5% Triton X-100 before PFA fixation. Incubation with primary antibodies against PR-Set7 (1:600; Millipore), HA tag (12CA5; 1:500), phospho-H2A.X-Ser139 (1:500; Millipore), phosphohistone H3-Ser10 (1:600; Millipore), proliferating cell nuclear antigen (1:500; gift from J. Piette), DNA polymerase
(1:800; gift from D. Fischer, Institut de Génétique Moléculaire, Montpellier, France), and 53BP1 (1:500; Millipore) was conducted at room temperature for 1 h. After washing, cells were incubated with AlexaFluor568- or 488-conjugated goat anti–mouse or goat anti–rabbit secondary antibodies (Invitrogen) for 1 h. Cells were finally stained with DAPI, and coverslips were mounted using Prolong Antifade (Invitrogen). Samples were examined at room temperature with a microscope (Imager.Z1; Carl Zeiss, Inc.) equipped with a 63× NA 1.4 or 40× NA 1.25 oil immersion objective and an Apotome device (Carl Zeiss, Inc.). Pictures were captured using a camera (CoolSNAP HQ2
; Photometrics) interfaced with MetaMorph software 7.1 (MDS Analytical Technologies), and levels were adjusted with Photoshop 7 (Adobe). Digital sectioning images (Apotome) were captured using Axiovision 4.6 software (Carl Zeiss, Inc.).
3 d after shPR7 or shLuc expression, HNFE6 cells were incubated for 30 min with 10 μg/ml colchicine. After washing in PBS, cells were resuspended in a hypotonic solution of 75 μM KCl for 15 min at 37°C. They were then fixed twice with a methanol/acetic acid buffer for 10 min at 4°C and spotted on frozen microscope glass slides. After drying, slides were stained with a solution of PBS and 0.1% Tween 20 containing Hoescht (1:10,000) mounted using Prolong Antifade, and images were captured at room temperature using an Imager.Z1 microscope with a 100× NA 1.4 oil immersion objective (Carl Zeiss, Inc.) equipped with a CoolSNAP HQ2 camera interfaced with MetaMorph software 7.1. Levels were adjusted with Photoshop 7.0.
U2OS cells were pulse labeled for 15 min with 50 μM BrdU and were harvested. For IdU-CldU labeling experiments, cells were successively labeled for 1 h with 25 μM IdU and 200 μM CldU for 1 h. Nuclei (from ~2.5 × 104
cells) were embedded in agarose plugs, stained with YOYO-1 (Invitrogen), and resuspended in 50 mM MES, pH 5.6, after digestion of the plugs with agarase (Roche). Then, DNA combing was performed as described previously (Michalet et al., 1997
). In brief, combed DNA fibers were denatured for 25 min with 0.2 N NaOH, and BrdU and CldU were detected with a rat anti-BrdU antibody (clone BU-75; Sera Laboratory) and a secondary antibody coupled to AlexaFluor488 (Invitrogen). IdU was detected with a mouse monoclonal anti-IdU antibody (clone B44; Becton Dickinson) and a secondary antibody coupled with AlexaFluor546 (Invitrogen). DNA molecules were counterstained as previously described (Pasero et al., 2002
) with an antiguanosine antibody (Argene) and an anti–mouse IgG coupled to AlexaFluor546 (Invitrogen). DNA fibers were examined, and images were captured at room temperature using a microscope (DMRX; Leica) with a 40× NA 1.25 oil immersion objective (Leica) and equipped with a CoolSNAP FX camera interfaced with MetaMorph software 7.1. Levels were adjusted with Photoshop 7.0. Replicated track sizes were measured with MetaMorph using adenovirus DNA molecules as a size standard (one pixel = 680 bp). Fork speed was calculated by dividing track sizes by labeling time, and the nonparametric test of Mann-Whitney was used.
DNA break repair was assayed by alkaline single-cell agarose gel electrophoresis essentially as described previously (Murr et al., 2006
). In brief, cells were infected with shRNA luc (control) or shRNA PR-Set7, harvested (~105
per pellet) 5 d after infection, mixed with low-gelling temperature agarose (type VII; Sigma-Aldrich), and layered onto agarose-coated glass slides. Slides were maintained in the dark at 4°C to gel and were submerged in lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Trizma base, 1% Triton X-100, and 10% DMSO) for at least 1.5 h. After washing with Tris buffer, slides were incubated for 1 h in alkaline electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 10) or in neutral electrophoresis buffer (300 mM sodium acetate, 100 mM Tris-HCl, and 1% DMSO, pH 8.3). Slides were then subjected to electrophoresis for ~40 min at 25 V, air dried, neutralized, and stained with 30 μl ethidium bromide (20 μg/ml−1
). Mean comet tail moment was scored for 50 cells/slide by using Comet Imager 1.2.10 software (MetaSystems Inc.).
Online supplemental material
Fig. S1 shows the cell cycle distribution of PR-Set7 in U2OS and normal fibroblasts (A) and in the nuclear localization of HA epitope–tagged PR-Set7 in U2OS cells treated for 1 h with proteasome inhibitor MG132 (B). Fig. S2 shows representative DNA combing pictures in shLuc and shPR7 U2OS cells. Fig. S3 shows 53BP1 localization at spontaneous DNA damage foci in shPR7-depleted U2OS cells and quantification of shPR7 U2OS cells displaying 53BP1 foci after ionizing radiation. Fig. S4 shows the impact of PR-Set7 depletion in proliferation rates and H4-K20me status of HeLa cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200706179/DC1